System and process for recovering algal oil

ABSTRACT

Herein disclosed is a method of processing a medium containing algae microorganisms to produce algal oil and by-products, comprising providing the medium containing algae microorganisms; passing the medium through a rotor-stator high shear device; disintegrating cell walls of and intracellular organelles in the algae microorganisms to release algal oil and by-products; and removing the algae medium from an outlet of the high shear device. In an embodiment, disintegration is enhanced by a penetrating gas capable of permeating the cell wall. In an embodiment, enhancement is accomplished by super-saturation of the penetrating gas in the medium or increased gas pressure in a vessel. In an embodiment, the penetrating gas is different from the gas produced by the cell during respiration. A suitable system is also discussed in this disclosure.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

BACKGROUND OF THE INVENTION

Technical Field

The present invention relates generally to recovery of algal oil. Moreparticularly, the present invention relates to a system and process forculture of algae and extraction of algal oil using high shear.

Background of the Invention

In recent years, algal oil has been recognized as a valuableagricultural land-saving alternative to plant feedstock, such as soybeanoil, canola and palm oil or animal, fish and bird fat and tallow. Algaeare the simplest plants that live in a water environment. Many algae areunicellular that may or may not have a cell wall. Similar to otherplants, algae are photosynthetic. They utilize carbon dioxide as acarbon source and store energy in the form of lipids within theintracellular oil bodies, surrounded by membranes. Algae multiply andgrow at a very fast rate and, depending on the genetic background andgrowth conditions, may have very high oil content. In biodieselproduction, the wild type strains of algae or mutants and geneticallymodified microorganisms, which are designed and selected to produceenhanced levels of oil and/or high levels of oleic acid, are preferred.Such strains can be obtained by Polymerase Chain Reaction (PCR)mutagenesis or by exposure to ultraviolet or ionizing radiation andchemical mutagens. Oil-overproducing strains can be engineered with thehelp of the directed evolution and other biotechnological techniquesknown to those skilled in the art.

In addition to methods and systems that stimulate and increaseproduction of oil in algae during culture, there is also the need formethods and systems to extract algal oil. There are a number of methodsfor disintegration of algae and recovering the algal oil, such aspressing, extraction with organic solvents, enzymatic degradation, lysisusing osmosis and ultrasonic and microwave-assisted disruption. Some ofthe new technologies for algal oil extraction include enzymatichydrolysis, pulsed electric field (PEF) technology, and the use ofamphyphillic solvents.

Clearly, there is a need and interest to continue to develop systems andmethods for algal oil extraction. Preferably such systems and methodsare economical and able to handle large volume of algal culture.

SUMMARY

Herein disclosed is a method of processing a medium containing algaemicroorganisms to produce algal oil and by-products, comprisingproviding the medium containing algae microorganisms; passing the mediumthrough a rotor-stator high shear device; disintegrating cell walls ofand intracellular organelles in the algae microorganisms to releasealgal oil and by-products; and removing the algae medium from an outletof the high shear device. In an embodiment, disintegration is enhancedby a penetrating gas capable of permeating the cell wall. In anembodiment, enhancement is accomplished by super-saturation of thepenetrating gas in the medium or increased gas pressure in a vessel. Inan embodiment, the penetrating gas is different from the gas produced bythe cell during respiration.

In an embodiment, the medium containing algae microorganisms isde-watered at least partially before the medium is passed through thehigh shear device. In an embodiment, a solvent is added to the at leastpartially de-watered medium before the medium is passed through the highshear device. In an embodiment, the solvent is a gas comprising carbondioxide or air or oxygen or nitrogen; or a liquid comprising an alcoholor hexane or vegetable oil and/or animal fat or tallow.

In an embodiment, the method comprises separating algal oil andbyproducts from the algae medium removed from the high shear device. Inan embodiment, the method comprises converting the algal oil tobiodiesel.

In an embodiment, the method comprises producing the medium containingalgae microorganisms, comprising super-saturating a liquid with carbondioxide in a second rotor-stator high shear device operating at a shearrate of greater than 1,000,000 s⁻¹; feeding the carbon dioxidesupersaturated liquid and a nutrient source to algae microorganisms andoptionally bacteria; allowing the algae microorganisms to grow byconsuming carbon dioxide and the nutrient; and generating the mediumcontaining algae microorganisms.

In an embodiment, the nutrient source comprises municipal waste; sewagewaste; paper pulp; chemical and petrochemical; vegetable includinggrain, sugar; farm discharge; animal farm discharge including beef,pork, poultry; canning discharge, fishing discharge; farming discharge;food processing discharge. In an embodiment, the nutrient source ispretreated to eliminate undesirable pathogens via gas-assisted highshear lysing of pathogen cells or pretreated using high shear toincrease the bio-availability of nutrient in the nutrient source. In anembodiment, the algae and/bacteria are genetically modified. In anembodiment, the bacteria cause the breakdown of the nutrient source topromote algae growth.

Herein also disclosed is a system comprising a rotor-stator high sheardevice configured to process a medium containing algae microorganisms toproduce algal oil and by-products, wherein the high shear device isoperated to disintegrate cell walls of and intracellular organelles inthe algae microorganisms to release algal oil and by-products, whereinthe high shear device comprises an inlet to take in the mediumcontaining algae microorganisms and an outlet for the algae medium to beremoved from the high shear device.

In an embodiment, the system comprises at least two rotor-stator highshear devices fluidly connected in series to process the mediumcontaining algae microorganisms and disintegrate cell walls of andintracellular organelles in the algae microorganisms to release algaloil and by-products. In an embodiment, the system comprises a separationsystem configured to separate algal oil and by-products from the medium.In an embodiment, the system comprises a conversion system configured toconvert algal oil to biodiesel.

In an embodiment, the system comprises a tank or pond configured to growalgae containing algae microorganisms and optionally bacteria; anutrient source consumable by the algae microorganisms and optionallybacteria; another rotor-stator high shear device configured to processcarbon dioxide in a liquid operating at a shear rate of greater than1,000,000 s−1 to form a carbon dioxide super-saturated liquid stream andfeed the stream into the tank/pond for algae growth; and a fluid lineconfigured to extract a medium containing algae from the tank or pondand send the medium to the rotor-stator high shear device configured toprocess the medium.

In an embodiment, the system of this disclosure is modular and isoptionally integrated with an existing facility.

Certain embodiments of the above-described methods or systemspotentially provide overall cost reduction by providing increasedinhibition per unit of inhibitor consumed, permitting increased fluidthroughput, permitting operation at lower temperature and/or pressure,and/or reducing capital and/or operating costs. These and otherembodiments and potential advantages will be apparent in the followingdetailed description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more detailed description of the preferred embodiment of thepresent invention, reference will now be made to the accompanyingdrawings, wherein:

FIG. 1 is a longitudinal cross-section view of a multi-stage high sheardevice, as employed in an embodiment of the system.

FIG. 2 is an overall process flow diagram for algae culture and algaloil recovery, according to an embodiment of this disclosure.

FIG. 3 illustrates the super saturation process of the feedstock foralgae, according to an embodiment of this disclosure.

FIG. 4 illustrates a process for algae lysing and algal oil recovery,according to an embodiment of this disclosure.

FIG. 5 illustrates another process for algae lysing and algal oilrecovery, according to an embodiment of this disclosure.

FIG. 6 illustrates detail ‘A’ for fluid connections as shown in FIGS. 4,5 and 11, according to an embodiment of this disclosure.

FIGS. 7 and 8 show dissolved oxygen concentration of a 1:10supersaturated oxygen solution in Example 1.

FIG. 9 shows dissolved carbon concentration of a shear-induced, CO₂infused distilled water solution over time in Example 1.

FIG. 10 shows the percentage of dissolved carbon remaining in solutionover time in Example 1, which is independent of SSCO2 concentration.

FIG. 11 illustrates a CO2-assisted high shear algae cell disruption flowdiagram, according to an embodiment of this disclosure.

FIG. 12 shows the particulates observed within the rotor of thehigh-shear unit in Example 2.

FIG. 13 shows time dependent effects of SSCO2-assisted high shear lysisof algae cells observed by microscopy in Example 2.

FIG. 14 shows representative images of processed algae slurry throughthe CO2-assisted high shear unit in Example 2.

FIG. 15 shows representative microscopic images of control and processed(SSCO2-shear-colloid mill) algae slurry in Example 2.

FIG. 16 shows the oily surface observed on CO2-assisted high shearprocessed algae slurry in Example 2.

FIG. 17 illustrates excess O2 leading to reactive oxygen species (ROS)generation and cell death within yeast cells in Example 3.

FIG. 18 shows representative microscopic images (40×) of unprocessedcontrol yeast (A), 1st pass samples exposed to 0 psi back pressure (B),20 psi back pressure (C), and 40 psi back pressure (D) in Example 3.

FIG. 19 shows representative microscopic images (40×) of unprocessedcontrol yeast (A), 2nd pass samples exposed to 20 psi back pressure (B),and 40 psi back pressure (C) in Example 3.

FIG. 20 shows, in Example 3, representative microscopic images (40×) ofyeast continuously circulated through the O2-assisted shear (HSPD+CM)system for 5 minutes. (A) unprocessed (circulated, no O2, no high shearprocessing unit, no colloidal mill) control yeast, (B) high shearprocessing unit+colloidal mill (no O2), (C) high shear processingunit+colloidal mill, 20 psi back pressure, (D) high shear processingunit+colloidal mill, 40 psi back pressure.

FIG. 21 illustrates a process flow diagram of the cell lysis method andsystem as discussed in Example 4.

FIG. 22 illustrates the effects of high shear speed on yeast cell lysisassisted by O2 as discussed in Example 4.

DETAILED DESCRIPTION

It has been unexpectedly discovered that the use of a rotor-stator highshear device is effective in the disintegration of unicellular and/ormulticellular algal (and/or bacterial) microorganisms and theirintracellular organelles to release oil and other cell contents. Afterlysing the algal (and/or bacterial) microorganisms and theirintracellular organelles, the product is separated and converted tomodified, more valuable products, e.g. algal oil. This finding iscontrary to the general knowledge and wisdom of the field of art. Themethod and system of this application also bypass the cost for expensiveapparatus and have low operational cost. Examples of rotor-stator highshear device include mechanical pulpers, refiners (e.g., Beloit Jones DD3000 refiners, Voith Twin Flo TFE Refiner, Metso JC series refiners),mills. Papermaking refiners can be either disc refiners or conicalrefiners. The pulp enters through a feed port, travels between a conicalrotor and stator and then leaves through the discharge port. The rotorand stator will have a bar and groove pattern. Only one of the elementswill rotate (the rotor). The gap between the refiners can be controlledby pushing the rotor and stator together. A disc refiner is very similarto the conical refiner. The pulp travels between two discs with bars andgrooves. There are essentially three categories of disc refiner:

1. Single disc refiners, where the pulp goes between a rotating rotorand a stationary stator.2. Twin refiner where the rotor and stator both rotate.

In papermaking the refiner serves to increase the flexibility of thecell wall in order to promote increased contact area, and also tofibrillate the external surface of the cell wall to further promote theformation of hydrogen bonds as well as increase the total surface areaof fiber available for bonding.

The present invention can utilize any of the rotor stator designs andconfigurations to create high shear for the purpose of enhancing cellgrowth through gas super saturation and lysing cells.

As used herein the term supersaturating of gas is used to describe gasheld within a liquid whose instantaneous quantity exceeds that expectedthrough Henry's Law. Although not wanting to be limited by theory,supersaturation of gases is believed to occur due to the extremepressures under high shear conditions that increases the solubility ofthe gas combined with micro dispersion of the gases in the liquid.

Furthermore, it has been unexpectedly discovered that lysis of cells isenhanced by using a high shear device coupled with introduction of a gascapable of permeating the cell wall and thus expanding the cell andreducing the cell wall integrity. Such enhancement is achieved by highshear coupled with gas super-saturation in the culture medium duringlysing or high shear coupled with a gas-pressurized vessel. The highshear device may be of a rotor-stator design. Migration of gas isenhanced through super saturation of gases by means of high shear or viaincreased pressures such as in a vessel under elevated pressure. Varioustechniques that utilize pressure to enhance cell lysing are known tothose experienced in the art including what is commonly referred to as aFrench Press.

In an embodiment, enhancing cell lysing involves selection of a suitablegas that preferentially penetrates cell walls. The selection of asuitable gas is dependent on the nature of the cell and the gas producedby the cell during respiration. As an example, yeast that produce carbondioxide did not exhibit enhanced lysing when exposed to supersaturatedcarbon dioxide while yeast exposed to supersaturated oxygen did exhibitenhanced lysing. The enhancement of yeast lysing while exposed tosupersaturated oxygen was attributed to the differential gas pressureacross the cell wall and the observed expansion of the cell underexposure to oxygen as opposed to carbon dioxide.

The disclosed method and system of enhanced cell lysing can be appliedto any gas producing algae. Selection of a suitable supersaturated gasis dependent on the nature of the gas being produced by the cell duringrespiration. Thus, a cell such as algae producing oxygen and would beexpected to have a higher concentration of oxygen within the cell wallwould not be expected to experience enhanced lysing from exposure tosupersaturated oxygen. Gases such as carbon dioxide, nitrogen, sulfuroxides, and other gases that would expect to migrate from the medium towithin the cell walls and reduce cell wall integrity would be expectedto enhance lysing. Similarly it has been shown that yeast producingcarbon dioxide did not experience enhanced lysing when exposed tosupersaturated carbon dioxide but did exhibit enhanced lysing whenexposed to supersaturated oxygen.

Although oxygen, nitrogen carbon dioxide and sulfur oxide have beennoted as gases to be used in enhancing cell lysing, other suitable gasescan be used to obtain the same effect of reducing cell wall integrity,e.g., hydrogen, methane. In an embodiment, hydrogen is super saturatedinto a medium and contributes to enhanced lysing in cells that are notproducing hydrogen during respiration.

High Shear Device.

The high shear device of this application is shown in FIG. 1 anddescribed herein. Although only one high shear device is shown in FIG.1, it should be understood that some embodiments of the system may havetwo or more high shear devices arranged either in series or parallelflow. HSD 200 is a mechanical device that utilizes one or more generatorcomprising a rotor/stator combination, each of which has a gap betweenthe stator and rotor. The gap between the rotor and the stator in eachgenerator set may be fixed or may be adjustable. HSD 200 is configuredin such a way that it is capable of producing submicron and micron-sizedbubbles or droplets of inhibitor in a continuous phase comprising thecarrier flowing through the high shear device. The high shear devicecomprises an enclosure or housing so that the pressure and temperatureof the fluid therein may be controlled.

High shear devices are generally divided into three general classes,based upon their ability to mix fluids. Mixing is the process ofreducing the size of particles or inhomogeneous species within thefluid. One metric for the degree or thoroughness of mixing is the energydensity per unit volume that the mixing device generates to disrupt thefluid particles. The classes are distinguished based on delivered energydensities. Three classes of industrial mixers having sufficient energydensity to consistently produce mixtures or emulsions with particlesizes in the range of submicron to 50 microns include homogenizationvalve systems, colloid mills and high speed mixers. In the first classof high energy devices, referred to as homogenization valve systems,fluid to be processed is pumped under very high pressure through anarrow-gap valve into a lower pressure environment. The pressuregradients across the valve and the resulting turbulence and cavitationact to break-up any particles in the fluid. These valve systems are mostcommonly used in milk homogenization and can yield average particlesizes in the submicron to about 1 micron range. The high shear devicealso includes attrition mills.

At the opposite end of the energy density spectrum is the third class ofdevices referred to as low energy devices. These systems usually havepaddles or fluid rotors that turn at high speed in a reservoir of fluidto be processed, which in many of the more common applications is a foodproduct. These low energy systems are customarily used when averageparticle sizes of greater than 20 microns are acceptable in theprocessed fluid.

Between the low energy devices and homogenization valve systems, interms of the mixing energy density delivered to the fluid, are colloidmills and other high speed rotor-stator devices, which are classified asintermediate energy devices. A typical colloid mill configurationincludes a conical or disk rotor that is separated from a complementary,liquid-cooled stator by a closely-controlled rotor-stator gap, which iscommonly between 0.025 mm to 10 mm (0.001-0.40 inch). Rotors are usuallydriven by an electric motor through a direct drive or belt mechanism. Asthe rotor rotates at high rates, it pumps fluid between the outersurface of the rotor and the inner surface of the stator, and shearforces generated in the gap process the fluid. Many colloid mills withproper adjustment achieve average particle sizes of 0.1-25 microns inthe processed fluid. These capabilities render colloid mills appropriatefor a variety of applications including colloid and oil/water-basedemulsion processing such as that required for cosmetics, mayonnaise, orsilicone/silver amalgam formation, to roofing-tar mixing.

Tip speed is the circumferential distance traveled by the tip of therotor per unit of time. Tip speed is thus a function of the rotordiameter and the rotational frequency. Tip speed (in meters per minute,for example) may be calculated by multiplying the circumferentialdistance transcribed by the rotor tip, 27 a, where R is the radius ofthe rotor (meters, for example) times the frequency of revolution (forexample revolutions per minute, rpm). A colloid mill, for example, mayhave a tip speed in excess of 22.9 m/s (4500 ft/min) and may exceed 40m/s (7900 ft/min). For the purpose of this disclosure, the term ‘highshear’ refers to mechanical rotor stator devices (e.g., colloid mills orrotor-stator dispersers) that are capable of tip speeds in excess of 5.1m/s. (1000 ft/min) and require an external mechanically driven powerdevice to drive energy into the stream of products to be reacted. Forexample, in HSD 200, a tip speed in excess of 22.9 m/s (4500 ft/min) isachievable, and may exceed 40 m/s (7900 ft/min). In some embodiments,HSD 200 is capable of delivering at least 300 L/h at a tip speed of atleast 22.9 m/s (4500 ft/min). The power consumption may be about 1.5 kW.HSD 200 combines high tip speed with a very small shear gap to producesignificant shear on the material being processed. The amount of shearwill also be dependent on the viscosity of the fluid in HSD 200.Accordingly, a local region of elevated pressure and temperature iscreated at the tip of the rotor during operation of the high sheardevice. In some cases the locally elevated pressure is about 1034.2 MPa(150,000 psi). In some cases the locally elevated temperature is about500° C. In some cases, these local pressure and temperature elevationsmay persist for nano or pico seconds.

An approximation of energy input into the fluid (kW/L/min) can beestimated by measuring the motor energy (kW) and fluid output (L/min).As mentioned above, tip speed is the velocity (ft/min or m/s) associatedwith the end of the one or more revolving elements that is creating themechanical force applied to the fluid. In embodiments, the energyexpenditure of HSD 200 is greater than 1000 watts per cubic meter offluid therein. In embodiments, the energy expenditure of HSD 200 is inthe range of from about 3000 W/m³ to about 7500 W/m³.

The shear rate is the tip speed divided by the shear gap width (minimalclearance between the rotor and stator). The shear rate generated in HSD200 may be in the greater than 20,000 s⁻¹. As used herein the term s⁻¹defined as inverse seconds, a term known to those experienced in the artto be used in defining shear rate for a fluid flowing between twoparallel plates In some embodiments the shear rate is at least 40,000s⁻¹. In some embodiments the shear rate is at least 100,000 s⁻¹. In someembodiments the shear rate is at least 500,000 s⁻¹. In some embodimentsthe shear rate is at least 1,000,000 s⁻¹. In some embodiments the shearrate is at least 1,600,000 s⁻¹. In some embodiments the shear rate is atleast 2,000,000 s⁻¹. At high shear rates (e.g., above 1,000,000 s⁻¹ or1,600,000 s⁻¹ or 2,000,000 s⁻¹), the HSD is able to super-saturate theliquid/medium with a gas (or gases), which is advantageous for algalculture wherein it is necessary and desirable to deliver highconcentrations of CO₂ to algae. So far, such delivery has been abottleneck for algal culture and thus for deriving algal oil. Algae arediverse group of photosynthetic organisms that typically grow in bodiesof water as unicellular or multicellular forms. As aquatic or marineorganisms, algae acquire the carbon dioxide necessary for photosynthesisby Brownian motion and diffusion. Further, certain species of algae fixcarbon derived from carbon dioxide to produce and store fatty oils,carbohydrates, proteins, polysaccharides, and other compounds,hereinafter hydrocarbons. The acquisition of carbon dioxide, hereinafterCO₂, from water represents a limiting step in growth rate and storage ofthese compounds. As certain algae are potentially useable in liquid fuelproduction, the uptake and fixation of carbon is a limiting step inpreparing alga-derived biofuels. The method and system of thisapplication are capable to reducing this hindrance and significantlyimprove efficiency for algal culture and algal oil recovery.

In embodiments, the shear rate generated by HSD 200 is in the range offrom 20,000 s⁻¹ to 100,000 s⁻¹. For example, in one application therotor tip speed is about 40 m/s (7900 ft/min) and the shear gap width is0.0254 mm (0.001 inch), producing a shear rate of 1,600,000 s⁻¹. Inanother application the rotor tip speed is about 22.9 m/s (4500 ft/min)and the shear gap width is 0.0254 mm (0.001 inch), producing a shearrate of about 901,600 s⁻¹. HSD 200 is capable of highly dispersing theinhibitor into a continuous phase comprising the carrier, with which itwould normally be immiscible. In some embodiments, HSD 200 comprises acolloid mill. Suitable colloidal mills are manufactured by IKA® Works,Inc. Wilmington, N.C. and APV North America, Inc. Wilmington, Mass., forexample. In some instances, HSD 200 comprises the Dispax Reactor® ofIKA® Works, Inc.

The high shear device comprises at least one revolving element thatcreates the mechanical force applied to the fluid therein. The highshear device comprises at least one stator and at least one rotorseparated by a clearance. For example, the rotors may be conical or diskshaped and may be separated from a complementarily-shaped stator. Inembodiments, both the rotor and stator comprise a plurality ofcircumferentially-spaced teeth. In some embodiments, the stator(s) areadjustable to obtain the desired shear gap between the rotor and thestator of each generator (rotor/stator set). Grooves between the teethof the rotor and/or stator may alternate direction in alternate stagesfor increased turbulence. Each generator may be driven by any suitabledrive system configured for providing the necessary rotation.

In some embodiments, the minimum clearance (shear gap width) between thestator and the rotor is in the range of from about 0.025 mm (0.001 inch)to about 3 mm (0.125 inch). In certain embodiments, the minimumclearance (shear gap width) between the stator and rotor is about 1.5 mm(0.06 inch). In certain configurations, the minimum clearance (sheargap) between the rotor and stator is at least 1.7 mm (0.07 inch). Theshear rate produced by the high shear device may vary with longitudinalposition along the flow pathway. In some embodiments, the rotor is setto rotate at a speed commensurate with the diameter of the rotor and thedesired tip speed. In some embodiments, the high shear device has afixed clearance (shear gap width) between the stator and rotor.Alternatively, the high shear device has adjustable clearance (shear gapwidth).

In some embodiments, HSD comprises a single stage dispersing chamber(i.e., a single rotor/stator combination, a single generator). In someembodiments, high shear device 200 is a multiple stage inline disperserand comprises a plurality of generators. In certain embodiments, HSD 200comprises at least two generators. In other embodiments, high sheardevice 200 comprises at least 3 high shear generators. In someembodiments, high shear device 200 is a multistage mixer whereby theshear rate (which, as mentioned above, varies proportionately with tipspeed and inversely with rotor/stator gap width) varies withlongitudinal position along the flow pathway, as further describedherein below.

In some embodiments, each stage of the external high shear device hasinterchangeable mixing tools, offering flexibility. For example, the DR2000/4 Dispax Reactor® of IKA® Works, Inc. Wilmington, N.C. and APVNorth America, Inc. Wilmington, Mass., comprises a three stagedispersing module. This module may comprise up to three rotor/statorcombinations (generators), with choice of fine, medium, coarse, andsuper-fine for each stage. This allows for creation of dispersionshaving a narrow distribution of the desired bubble or droplet size(e.g., gas bubbles or liquid droplets of inhibitor). In someembodiments, each of the stages is operated with super-fine generator.In some embodiments, at least one of the generator sets has arotor/stator minimum clearance (shear gap width) of greater than about 5mm (0.2 inch). In alternative embodiments, at least one of the generatorsets has a minimum rotor/stator clearance of greater than about 1.7 mm(0.07 inch).

Referring now to FIG. 1, there is presented a longitudinal cross-sectionof a suitable high shear device 200. High shear device 200 of FIG. 1 isa dispersing device comprising three stages or rotor-statorcombinations. High shear device 200 is a dispersing device comprisingthree stages or rotor-stator combinations, 220, 230, and 240. Therotor-stator combinations may be known as generators 220, 230, 240 orstages without limitation. Three rotor/stator sets or generators 220,230, and 240 are aligned in series along drive shaft 250.

First generator 220 comprises rotor 222 and stator 227. Second generator230 comprises rotor 223, and stator 228. Third generator 240 comprisesrotor 224 and stator 229. For each generator the rotor is rotatablydriven by input 250 and rotates about axis 260 as indicated by arrow265. The direction of rotation may be opposite that shown by arrow 265(e.g., clockwise or counterclockwise about axis of rotation 260).Stators 227, 228, and 229 may be fixably coupled to the wall 255 of highshear device 200.

As mentioned hereinabove, each generator has a shear gap width which isthe minimum distance between the rotor and the stator. In the embodimentof FIG. 1, first generator 220 comprises a first shear gap 225; secondgenerator 230 comprises a second shear gap 235; and third generator 240comprises a third shear gap 245. In embodiments, shear gaps 225, 235,245 have widths in the range of from about 0.025 mm to about 10 mm.Alternatively, the process comprises utilization of a high shear device200 wherein the gaps 225, 235, 245 have a width in the range of fromabout 0.5 mm to about 2.5 mm. In certain instances the shear gap widthis maintained at about 1.5 mm. Alternatively, the width of shear gaps225, 235, 245 are different for generators 220, 230, 240. In certaininstances, the width of shear gap 225 of first generator 220 is greaterthan the width of shear gap 235 of second generator 230, which is inturn greater than the width of shear gap 245 of third generator 240. Asmentioned above, the generators of each stage may be interchangeable,offering flexibility. High shear device 200 may be configured so thatthe shear rate will increase stepwise longitudinally along the directionof the flow 260.

Generators 220, 230, and 240 may comprise a coarse, medium, fine, andsuper-fine characterization. Rotors 222, 223, and 224 and stators 227,228, and 229 may be toothed designs. Each generator may comprise two ormore sets of rotor-stator teeth. In embodiments, rotors 222, 223, and224 comprise more than 10 rotor teeth circumferentially spaced about thecircumference of each rotor. In embodiments, stators 227, 228, and 229comprise more than ten stator teeth circumferentially spaced about thecircumference of each stator In embodiments, the inner diameter of therotor is about 12 cm. In embodiments, the diameter of the rotor is about6 cm. In embodiments, the outer diameter of the stator is about 15 cm.In embodiments, the diameter of the stator is about 6.4 cm. In someembodiments the rotors are 60 mm and the stators are 64 mm in diameter,providing a clearance of about 4 mm. In certain embodiments, each ofthree stages is operated with a super-fine generator, comprising a sheargap of between about 0.025 mm and about 4 mm.

High shear device 200 is configured for receiving at inlet 205 a fluidmixture from line 13. The mixture comprises inhibitor as the dispersiblephase and carrier fluid as the continuous phase. Feed stream enteringinlet 205 is pumped serially through generators 220, 230, and then 240,such that product dispersion is formed. Product dispersion exits highshear device 200 via outlet 210 (and line 18 of FIG. 1). The rotors 222,223, 224 of each generator rotate at high speed relative to the fixedstators 227, 228, 229, providing a high shear rate. The rotation of therotors pumps fluid, such as the feed stream entering inlet 205,outwardly through the shear gaps (and, if present, through the spacesbetween the rotor teeth and the spaces between the stator teeth),creating a localized high shear condition. High shear forces exerted onfluid in shear gaps 225, 235, and 245 (and, when present, in the gapsbetween the rotor teeth and the stator teeth) through which fluid flowsprocess the fluid and create product dispersion. Product dispersionexits high shear device 200 via high shear outlet 210.

The product dispersion has an average droplet or gas bubble size lessthan about 5 μm. In embodiments, HSD 200 produces a dispersion having amean droplet or bubble size of less than about 1.5 μm). In embodiments,HSD 200 produces a dispersion having a mean droplet or bubble size ofless than 1 μm; preferably the droplets or bubbles are sub-micron indiameter. In certain instances, the average droplet or bubble size isfrom about 0.1 μm to about 1.0 μm. In embodiments, HSD 200 produces adispersion having a mean droplet or bubble size of less than 400 nm. Inembodiments, HSD 200 produces a dispersion having a mean droplet orbubble size of less than 100 nm. The dispersion may be capable ofremaining dispersed at atmospheric pressure for at least about 15minutes.

Without wishing to be limited to a particular theory, it is believedthat the level or degree of high shear may be sufficient to increaserates of mass transfer and also produce localized non-ideal conditionsthat enable reactions to occur that would not otherwise be expected tooccur based on Gibbs free energy predictions. Localized non idealconditions are believed to occur within the high shear device resultingin increased temperatures and pressures with the most significantincrease believed to be in localized pressures. The increase inpressures and temperatures within the high shear device areinstantaneous and localized and quickly revert back to bulk or averagesystem conditions once exiting the high shear device. In some cases, thehigh shear device induces cavitation of sufficient intensity todissociate one or more of the reactants into free radicals, which mayintensify a chemical reaction or allow a reaction to take place at lessstringent conditions than might otherwise be required. Cavitation mayalso increase rates of transport processes by producing local turbulenceand liquid micro-circulation (acoustic streaming). An overview of theapplication of cavitation phenomenon in chemical/physical processingapplications is provided by Gogate et al., “Cavitation: A technology onthe horizon,” Current Science 91 (No. 1): 35-46 (2006). The high sheardevice of certain embodiments of the present system and methods inducescavitation whereby inhibitor and/or carrier fluid are dissociated intofree radicals. The cavitation effects cause and promote thedisintegration of algal cells, allowing fast and efficient release ofall intracellular oil.

Algal oil recovery process.

In an embodiment, the method of this disclosure comprises processing amedium containing algae microorganisms to produce algal oil andby-products, comprising providing the medium containing algaemicroorganisms; passing said medium through a rotor-stator high sheardevice; disintegrating cell walls of and intracellular organelles in thealgae microorganisms to release algal oil and by-products; and removingthe algae medium from an outlet of the high shear device. The processedmedium contains algal oil, extracted from the broken cells, the celldebris and other components. In some cases, the high shear device forcell lysing is operated at a shear rate of 20,000 to 10,000,000, oralternatively 100,000 to 2,000,000, or alternatively 200,000 to 500,000(in inverse seconds, s⁻¹). In some cases, the high shear device for celllysing is operated at a shear rate of 200,000 s⁻¹ or above. In somecases, the high shear device for cell lysing is operated at a shear rateof 200,000 s⁻¹ or above. In some cases, the high shear device for celllysing is operated at a shear rate of 300,000 s⁻¹ or above. In somecases, the high shear device for cell lysing is operated at a shear rateof 400,000 s⁻¹ or above. In some cases, the high shear device for celllysing is operated at a shear rate of 500,000 s⁻¹ or above. In somecases, the high shear device for cell lysing is operated at a shear rateof 600,000 s⁻¹ or above. In some cases, the high shear device for celllysing is operated at a shear rate of 700,000 s⁻¹ or above. In somecases, the high shear device for cell lysing is operated at a shear rateof 800,000 s⁻¹ or above. In some cases, the high shear device for celllysing is operated at a shear rate of 900,000 s⁻¹ or above. In somecases, the high shear device for cell lysing is operated at a shear rateof 1,000,000 s⁻¹ or above. In some cases, the high shear device for celllysing is operated at a shear rate of 2,000,000 s⁻¹ or above. It shouldbe noted that as the shear rate is calculated by measuring the shear gapand rotor speed, in the case where the configuration of the rotor statoris a disc or conical the shear rate (inverse seconds) will vary acrossthe disc or cone as the apparent rotational speed will vary across theradius of the disc. In such cases, the ranges mentioned above apply tothe average shear rates of such rotor-stator devices.

In some cases, the medium containing algae microorganisms is de-wateredat least partially before the medium is passed through said high sheardevice. In some cases, the medium is de-watered to obtain dry algae forfurther processing. In some embodiments, a solvent is added to the atleast partially de-watered medium before the medium is passed throughsaid high shear device. In some cases, the solvent is a gas comprisingcarbon dioxide or air or oxygen or nitrogen. In some cases, the solventis a liquid comprising an alcohol or hexane or vegetable oil and/oranimal fat or tallow. In an embodiment, 30% or more of algal oil isreleased for recovery.

After lysing the algae, the algal oil and byproducts are separated fromthe medium. The separation process is known in the art and comprises aprocess selected from the group consisting of washing, sedimentation,centrifugation, filtration, vaporization, distillation, freezing,extraction, or combinations thereof. Any suitable separation process iscontemplated to be within the scope of this disclosure. In embodiments,the separated algal oil is then converted to valuable products, e.g.,fuel including biodiesel, by any means known in the art. In embodiments,the separated byproducts are used for producing pharmaceuticals orfertilizers or animal feeds.

In an embodiment, 30% or more of algal oil is released and recovered. Inan embodiment, 40% or more of algal oil is released and recovered. In anembodiment, 40% or more of algal oil is released and recovered. In anembodiment, 50% or more of algal oil is released and recovered. In anembodiment, 60% or more of algal oil is released and recovered.

In an embodiment, 70% or more of algal oil is released and recovered. Inan embodiment, 80% or more of algal oil is released and recovered. In anembodiment, 90% or more of algal oil is released and recovered.

Algae Culture Process.

In a further embodiment, an improved algae culture/growth processcomprises super-saturating a liquid with carbon dioxide in a secondrotor-stator high shear device operating at a shear rate of greater than1,000,000 s⁻¹; feeding said carbon dioxide supersaturated liquid and anutrient source to algae microorganisms and optionally bacteria;allowing the algae microorganisms to grow by consuming carbon dioxideand the nutrient; and generating said medium containing algaemicroorganisms. In various embodiments, the nutrient source is anysource that enables microorganism growth, including but not limited tomunicipal waste, sewage waste, paper pulp, chemical and petrochemical,vegetable (grain, sugar), farm discharge, animal farm discharge (beef,pork, poultry), canning discharge, or fishing discharge as well as othernutrient containing discharge from other farming and farm productprocessing operations. Carbon dioxide may be obtained from any source,such as power plants, refineries, paper mills.

In various embodiments, the nutrient source may contain undesirablemicroorganisms, e.g., sewage waste. These undesirable microorganisms areherein called pathogens in this disclosure. As such, the nutrient ispretreated before fed to the microorganisms. The pretreatment compriseslysing the cells of the undesirable pathogens using a high shear deviceassisted by a penetrating gas (e.g., in super-saturation state) asdiscussed herein. Preferably, the penetrating gas is different from thegas produced by the cells of the pathogens during respiration. Forexample, the nutrient source is super saturated with a blend of oxygen(air or oxygen-enriched air or oxygen) and carbon dioxide using a highshear device and then passed through another high shear device. Or thenutrient source is pretreated in a high shear device while a suitablepenetrating gas or gas combination is simultaneously fed into the highshear device. Alternatively, the nutrient source is held in a FrenchPress containing a pressurized penetrating gas for a period of time andthen processed in a high shear device. By such pretreatment, thepathogens are eliminated and the nutrient source is suitable for algaeculture.

In other embodiments, the pretreatment of the nutrient source using highshear (with or without gas assistance) is able to break down thenutrient (e.g., sugar, carbohydrate) in the nutrient source to minisculesize so that it is easily digested/consumed by the microorganisms. Assuch, the bio-availability of the dispersed nutrient is significantlyincreased to promote microorganism growth.

In embodiments, the algae and/or bacteria used in this process aregenetically modified. Such genetic modification is performed based onthe type of the nutrient source to maximize algae growth. The bacteriathat are co-cultured with algae cause the breakdown of said nutrientsource to promote algae growth.

System for Algal Oil Recovery.

In an embodiment, a system that is capable of recovering algal oilcomprise a rotor-stator high shear device configured to process a mediumcontaining algae microorganisms to produce algal oil and by-products,wherein said high shear device is operated to disintegrate cell walls ofand intracellular organelles in the algae microorganisms to releasealgal oil and by-products, wherein said high shear device comprises aninlet to take in said medium containing algae microorganisms and anoutlet for the algae medium to be removed from the high shear device. Insome cases, the system comprises at least two rotor-stator high sheardevices fluidly connected in series to process said medium containingalgae microorganisms and disintegrate cell walls of and intracellularorganelles in the algae microorganisms to release algal oil andby-products. In various embodiments, the system further comprises aseparation system configured to separate algal oil and by-products fromthe medium. In embodiments, the system further comprises a conversionsystem configured to convert algal oil to biodiesel. Such separation andconversion systems are known in the art and all such suitable systemsare considered to be within the scope of this disclosure.

System for Algae Culture.

In another embodiment, an algae culture system upstream of the algal oilrecovery system comprises a tank or pond configured to grow algaecontaining algae microorganisms and optionally bacteria; a nutrientsource consumable by said algae microorganisms and optionally bacteria;another rotor-stator high shear device configured to process carbondioxide in a liquid operating at a shear rate of greater than 1,000,000s⁻¹ to form a carbon dioxide super-saturated liquid stream and feed saidstream into the tank/pond for algae growth; and a fluid line configuredto extract a medium containing algae from the tank or pond and send themedium to the rotor-stator high shear device configured to process saidmedium. The combination of the algae culture system and algal oilrecovery system are modular, versatile, and movable. As such, it is easyto integrate such systems with an existing facility for carbon dioxidesequestration, waste management, and bio-fuel production. As is clear toone skilled in the art, such integration has numerous benefits andfulfills a multitude of purposes.

Algae culture and algal oil recovery. In an embodiment, as illustratedschematically in FIG. 2, an overall process flow of algae culture andalgal oil recovery is shown. A waste stream or water source (i.e.,nutrient source) is super saturated with gas (CO₂ and optionally O₂/air)in a high shear device. Oxygen or air is added in some cases, e.g., theuse of sewage waste as the nutrient source so that the BOD and CODcontents would meet standards. The supersaturated stream is introducedto algae/bacteria growth chamber. After the desired growth of algae isachieved, a medium containing algae is processed in another rotor-statorhigh shear device as described herein above for cell lysing. Algal oiland byproducts are released and subsequently recovered.

In an embodiment, as shown in FIG. 3, a feedstock (e.g., feedstock fromwater source and/or recycle stream from a chemical plant, power plant,waste treatment, paper mill or other process operation producing wastestream) is fed through a pump to one or more high shear units. Gasinjection of CO₂ (and optionally O₂/air) may take place before or afterthe pump before the high shear units. A supersaturated stream isobtained and fed to the growth vessel (or raceway/pond) of algae. Insome cases, a portion of the supersaturated stream is recycled throughthe high shear units. In some cases, a portion of the medium in thegrowth vessel is recycled through the high shear units. When desiredgrowth of algae is reached, a medium containing algae is taken from thegrowth vessel to the lysing and recovery processes downstream.

In an embodiment, as illustrated by FIG. 4, a medium containing algae(grown/mature algae) and optionally diatomaceous earth is sent through apump (e.g., Cole Palmer pump) to one or more high shear devices for celllysing. Optionally a solvent is added (e.g., CO₂ or hexane) eitherbefore the pump or after the pump to be processed in the high sheardevice. In some cases, one of the high shear devices is a MK2000/4colloid mill by IKA. After cell lysing, the medium containing thereleased algal oil, byproducts, and cell debris is sent to a separationand recovery process.

In an embodiment, as illustrated by FIG. 4, a medium containing algae(grown/mature algae) and inorganics (e.g., diatomaceous earth) is sentthrough a pump (e.g., Ross pump) to one or more high shear devices forcell lysing. Optionally a solvent is added (e.g., CO₂ or hexane) eitherbefore the pump or after the pump to be processed in the high sheardevice. In some cases, one of the high shear devices is a MK2000/4colloid mill by IKA. In some cases, recycle streams are extracted andsent through one or more high shear units for multiple-pass operation.After cell lysing, the medium containing the released algal oil,byproducts, and cell debris is sent to a sludge separation tank(optional). A bottom stream from the separation tank is taken to recyclefor biomass, sludge, water feed, and/or sugar recovery. The top layerfrom the separation tank is extracted and separated (under optionalvacuum) into oil, water, and biomass. The water and biomass are recycledfor desired use and the oil is further processed and converted tobiodiesel.

Detail ‘A’ in FIGS. 4 and 5 for fluid connection is shown in FIG. 6. Asis known to one skilled in the art, any comparable arrangement may bemade to serve the same function and is contemplated to be within thescope of this disclosure.

Features.

The disclosed method and system are versatile, low in capital cost andoperational cost. Furthermore, the system is modular, making it easy tobe integrated into any existing facility or infrastructure, e.g., powerplants, sewage treatment plants, canning factories, food processingunits, etc.

The separated solids may be recycled and used for many other purposes,such as producing pharmaceuticals, fertilizers, animal feeds, etc.

Example 1 Effects of Super-Saturation Using High Shear

Carbon dioxide (CO₂) is readily soluble in water in the form of adissolved gas. Surface waters normally contain less than 10 ppm freecarbon dioxide, while some ground waters may easily exceed thatconcentration. Over the typical temperature range (0-30° C.), thesolubility is about 200 times that of oxygen. When CO2 reacts withwater, it immediately forms carbonic acid (H₂CO₃), which is relativelyunstable. This further dissociates to form bicarbonate (HCO₃ ⁻) andcarbonate (CO₃ ²⁻) ions.

Compared with oxygen, the estimation of carbon dioxide in water presentsmuch greater difficulties. Although pH is widely used to measure thepresence of carbonic acids and carbonates in solution, the presence ofcarbonate forming ions, including calcium, magnesium, and sodium mayinterfere with total dissolved carbon measurements.

The total inorganic carbon (TIC) or dissolved inorganic carbon (DIC) isthe sum of inorganic carbon species (including carbon dioxide, carbonicacid, bicarbonate anion, and carbonate) in a solution. It is customaryto express carbon dioxide and carbonic acid simultaneously as CO2*. TICis a key parameter when making measurements related to the pH of naturalaqueous systems, and carbon dioxide flux estimates:

TIC=[CO₂*]+[HCO₃ ⁻]+[CO₃ ²⁻]

where, TIC is the total inorganic carbon; [CO₂*] is the sum of carbondioxide and carbonic acid concentrations ([CO₂*]=[CO₂]+[H₂CO₃]); [HCO₃⁻] is the bicarbonate concentration; [CO₃ ²⁻] is the carbonateconcentration.

Each of these species are related by the following pH-driven chemicalequilibrium equation:

CO₂+H₂O

H₂CO₃

H⁺+HCO₃ ⁺

₂H⁺+CO₃ ²⁻

The concentrations of the different species of DIC (and which species isdominant) depend on the pH of the solution, as shown by a Bjerrum plot.Total inorganic carbon is typically measured by the acidification of thesample which drives the equilibria to CO₂. This gas is then sparged fromsolution and trapped, and the quantity trapped is then measured, usuallyby infrared spectroscopy using a Total Organic Carbon (TOC) analyzer.

Total Organic Carbon (TOC) is a sum measure of the concentration of allorganic carbon atoms covalently bonded in the organic molecules of agiven sample of water. TOC is typically measured in Parts Per Million(ppm or mg/L). As a sum measurement, Total Organic Carbon does notidentify specific organic contaminants. It will, however, detect thepresence of all carbon-bearing molecules, thus identifying the presenceof any organic contaminants, regardless of molecular make-up.

A typical analysis for TOC measures both the Total Carbon (TC) as wellas Inorganic Carbon (IC, or carbonate). Subtracting the Inorganic Carbonfrom the Total Carbon yields TOC. (TC−IC=TOC).

Dissolved oxygen can easily be measured and reported as mg/L using adissolved oxygen probe (Milwaukee Instruments) submerged in solution.

Initially, 3 L of distilled water were sheared using a high shear deviceto monitor the concentration of dissolved oxygen in solution over time.However, the concentration of dissolved oxygen exceeded the measurementcapability of the dissolved oxygen probe (MW600, Milwaukee Instruments).Therefore, the test was repeated and the supersaturated oxygen solutionwas immediately diluted 1:10 prior to measurements. In this case, threeliters of distilled water were sheared in the presence of oxygen gas andquickly diluted to a 1:10 concentration in 3 one liter flasks. Theconcentration of the 1:10 supersaturated oxygen solution was initiallymeasured to be an average of 17.6 mg/L. Within 5 minutes, theconcentration of the dissolved oxygen dropped to an average of 6.7 mg/L,a 62% loss of dissolved oxygen in the 1:10 solution (FIGS. 7 and 8). Toestablish the dissolved oxygen equilibrium point, the dissolved oxygenconcentration was monitored daily for a total of 4 days. The data showthat a dissolved oxygen equilibrium point was reached between 2 and 3days following supersaturation, and that the dissolved oxygenconcentration maintained a final concentration of 4.3 mg/L. As acontrol, the dissolved oxygen concentration of distilled water wasconcurrently monitored at 4.3 mg/L throughout the test. These resultsindicate that the tested shear technology effectively supersaturatesdistilled water with oxygen; however, this effect is of very shortduration.

Similar to the supersaturated oxygen tests, 3 liters of distilled waterwere sheared in the presence of carbon dioxide and evaluated for totaldissolved carbon using a TOC analyzer. Also like the dissolved oxygentests, the concentration of dissolved carbon in the neat solutionexceeded the instrument's measurement capability. Therefore, the neatsolution was diluted to 1:10, 1:100, and 1:1000 in triplicate toquantify dissolved carbon over time. Potassium hydrogen phthalate (KHP)is commonly used as a standard for dissolved carbon measurements by TOC.A freshly prepared 10 g/L KHP solution of was diluted into aconcentration range between 1 mg/L-1000 mg/L and used to establish thedissolved carbon standard curve. This range of standards was prepareddaily, while the original diluted supersaturated CO2 solutions weremaintained in septum-sealed glass vials and measured daily to quantifydissolved carbon over time and establish the SSCO2 equilibrium point.The pH of each solution was concurrently monitored.

The average initial concentration of the 1:10 dissolved carbon solutionwas measured to be 5675 ppm, or 5675 mg/L. The initial pH of eachsolution averaged 3.4. This unusually high concentration of dissolvedcarbon was confirmed by preparing fresh SSCO2 distilled water solutionsand repeating the TOC measurement. The concentration of dissolved carbondropped by about 47% within 2 days, then reached equilibrium within thefollowing 2 days (FIG. 9). The final dissolved carbon concentration ofthe 1:10 SSCO2 was measured to be an average of 2655 mg/L and a pH of3.4. As a control, distilled water was likewise measured. The TOCmeasurement of dissolved carbon in distilled water was 0 mg/L and the pHwas measured to be 7.

The concentration of dissolved carbon in the 1:100 and 1:1000 SSCO2solutions likewise diminished in the first 2-3 days, then reachedequilibrium within 4 days (FIG. 9). The pH of each of these solutionsalso remained consistent throughout the tests at pH 3.4 and 3.5respectively. These data indicate that even a small amount of dissolvedCO2 in distilled water causes a drop in pH from an initial value of 7 to3.4-3.5 and that pH alone cannot be used to calculate the concentrationof dissolved carbon in distilled water when excess carbon is present.Interestingly, when the loss of dissolved carbon is expressed as apercentage of the total measured carbon, all three dilutions showed asimilar pattern (FIG. 10). These data indicate that the rate of loss ofdissolved carbon from a shear-induced supersaturated solution isindependent of initial concentration and that the dilution of a SSCO2solution with additional distilled water may provide an accommodatingenvironment for excess CO2. It should also be noted these data werecollected from diluted solutions which may or may not be easilyextrapolated to reflect a neat SSCO2 solution.

Results Summary.

Shearing of distilled water in the presence of oxygen gas produces asupersaturated oxygen solution. Shearing of distilled water in thepresence of carbon dioxide gas produces a supersaturated carbon dioxidesolution that is maintained over time. The initial concentration ofdissolved carbon in a SSCO2 solution diluted to 1:10 was 5675 mg/L,which then equilibrated to maintain a concentration of 2655 mg/L. Thepercentage loss of dissolved carbon from diluted SSCO2 solutions wasconsistent over the range of dilutions. Addition of dissolved carbon todistilled water causes a drop in pH from 7 to −3.4, but does not reflectthe concentration of dissolved carbon when excess carbon is present.

As demonstrated by Example 1, the significant super-saturation level ofcarbon dioxide in solution caused by high shear processing is able topromote algae growth and reduce/resolve the bottleneck of insufficientCO₂ delivery to algae microorganisms, which is critical in algae cultureand recovery of algal oil.

Example 2 Effects of Cell Lysing Using High Shear

Cost-effective methods of disruption of the algal cell wall arefundamental to obtain higher lipid extraction efficiencies, meaninggreater net energy output from the process. For this example, theeffects of a method of algae cell disruption, CO2-assisted high shear,was assessed using cultured Chlorella sp. The combination of the actionsof supersaturated micro-sized CO₂ bubbles and physical shearing offer aunique hybrid approach to algae cell lysing and lipid recovery. It hasbeen unexpectedly discovered that supersaturated micro-sized CO₂ bubbleswork in synergy with mechanical high shear action to improve celldisintegration efficiency.

Although the energy required vs. energy recovery (energy return oninvestment) of this system was not assessed during the current testing,the system is expected to improve upon the EROI of other algaedisruption technologies because it does not require pre-drying, highpressures, nor increased heat. Rather, it relies on cost-effectivetechnologies. The efficiency and damage characteristics induced withthese treatments were quantified and evaluated using direct opticalmicroscopy and cell counting techniques. Release and recovery ofintracellular lipids were also quantified.

Materials and Methods. Algae Biomass and Lipid Production.

Chlorella sp. (UTEX 2714) were cultivated in Bold 3N growth media andscaled to 500 L within 8″ diameter vertical airlift photobioreactorsexposed to a daily 18/6 cycle of artificial illumination. Cell culturedensity was measured daily by dry cell weight. Nitrogen was limited oncethe culture reached 1.8 g/L to stimulate lipid accumulation within thecells. Once the culture density reached 2 g/L, 10% of each culture wastested as “dilute” culture while the remaining 90% of the culture wasconcentrated by centrifugation to a final “concentrate” of 12-15 g/Lpumpable slurry. Algae lipids were extracted using a modified Folchmethod, quantified gravimetrically, and expressed as % total lipid/drycell weight.

CO2-Assisted High Shear Algae Cell Disruption Set Up and Test Matrix.

The process flow for CO2-assisted high shear algae cell disruption isshown in FIG. 11. Biomass slurry sample flow and processing wasinitiated as follows: the fluid flow pressure of dilute or concentratedalgal slurry through the system was initiated at −80 psi andsubsequently increased to 85-105 psi. The high shear unit was thenturned on and maintained at a rotational speed of 15,000 or 26,000 rpmas indicated in the results section. Diffused CO2 was introduced intothe system at the shear unit an initial pressure of 80 psi, thenadjusted to 85-105 psi to reduce sample sputtering from the collectionnozzle. When applicable, the colloid mill was turned on. In some testcases, the back pressure to the shear unit was increased in incrementsof 20 psi from 0 psi to 80 psi. Once the target test parameters weremet, processed samples were collected in Erlenmeyer flasks forsubsequent cell disruption and lipid release analyses.

A matrix of test conditions were applied to the algae samples. Matrixvariables included fluid flow pressure entering the high shear unit (0,80-150 psi), back pressure to the high shear unit (0-80 psi), CO2pressure entering the high shear unit (0, 80-105 psi), and dilute (2 g/LDCW) vs. concentrated (12-13 g/L) Chlorella algae.

Quantification of Cell Disruption.

The effectiveness of each treatment was qualitatively visualized bybrightfield microscopy (10×-40×), and quantified by measuring thefraction of physically disrupted cells (Spiden, 2013). ImageJ, apublic-domain image processing and analysis software, was used to ensureconsistency in cell counting by minimizing variability in analysisbetween samples. Measurements of the total area occupied by cells wereused to verify the intact cell counts. This was particularly useful forsamples with cell clumping, typically observed in samples that had beenhomogenized at higher pressures. All imaging was performed using anAmScope B120C-E1 Siedentopf Binocular Compound Microscope with a 1.3 MPdigital camera. Cell counts were compared to unprocessed (flowed throughthe system but no shear, mill, or CO2) algae from the same batch. Cellviability was assessed using a standard XTT viability assay, whichmeasures mitochondrial enzymatic activity.

Quantification of Recovered Extracellular Lipids Following ShearProcessing.

Lipids released as a result of cell rupture were quantified by sweepingthe extracellular medium by inversion with 10% hexane for 30 seconds.The hexane layer containing released lipids was phase partitioned andrecovered following centrifugation of the sample. Following hexanedistillation, the recovered dry lipids were quantified gravimetricallyand expressed as the % of released lipids/dry cell weight of biomass.

System Operation and Cleaning.

Prior to activating the system for each set of tests, warm tap water wascycled through the system. Once flow was established, each component(high shear unit, CO2 gas flow, colloid mill) of the system was turnedon. The system was deemed fully operational if: 1) the fluid flowthrough the system was unrestricted, 2) the shear and colloid mill unitsreached full rotational speed, 3) all gas and fluid flow pressures wereachieved, and 4) the pH of the final water solution dropped to expectedlevels, typically from ˜pH 8-9 to ˜pH 5-6. The system was then turnedoff, the remaining water in the system was replaced with algae slurry,and the process was repeated prior to adjusting test parameters and datacollection. Following each set of tests, any remaining algae slurry inthe system was replaced with warm tap water, which was circulatedthrough the system for 30 minutes prior to shutting the system down. Insome cases, a thin opaque film lining the clear tubing in the system wasobserved in the following days, suggesting that a biofilm may havedeveloped within the system. This observation was further supported whenit was also noted that the fluid flow through the system appeared to beslightly restricted upon start-up. In these cases, isopropyl alcohol wascirculated through the system, followed by a citrate solution, until thefluid flow was restored.

Following system operation using diatomaceous earth, the restriction influid flow was even more pronounced and in some cases, the high shearunit became non-operational and displayed an Error F50 message (rotorshaft blocked). Cleaning the flow path with the isopropylalcohol-citrate method was insufficient to restore fluid flow or shearunit operation. Instead, the system required disassembly, direct softbrush cleaning, and reassembly before the system could be restored toand operational condition. The disassembly included the plumbingemanating from the mag lev pump to the shear unit and the rotor shaft ofthe high shear unit (FIG. 12). Once the system was manually cleaned andreassembled, operation was restored and testing was resumed. The issueswith restricted fluid flow caused by the addition of diatomaceous earthmay become less relevant as the system is scaled up to a commercialscale, although biofilm formation may still occur.

Results and Discussion.

In order to identify the operating conditions that maximized algae celldisruption, a test matrix in which individual parameters were varied oneat a time was created. Testing started with the basic CO2-assisted highshear system alone, followed by the addition of diatomaceous earth(silica remnants of algae diatoms) to enhance local shearing, andfinally by the downstream addition of a colloid mill.

CO2-Assisted High Shear Algae Cell Disruption.

Initially, dilute and concentrated Chlorella sp. cells were processedthrough the CO2-assisted high shear unit at 15,000 rpm withoutadditional exposure to the downstream colloid mill. In our previouswork, we had shown that the infusion of excess CO2 into the processstream supersaturates the effluent solution with CO2 and causes asignificant drop in slurry pH. Since the infusion of excess CO2 into theshear unit was integral to the current cell disruption process flow, weagain monitored pH throughout these tests. Along with changes in pH, thefluid flow pressures at various points throughout the process flow pathwere systematically changed one at a time in order to identify theoperating parameters that maximized algae cell rupture, which wasqualitatively evaluated microscopically following each sample run. Toquantify the percentage of cells disrupted (lysed) following some testruns, cell counts were also performed on equal volumes of processedunprocessed control cells from the same batch. The pH of the processedslurry dropped by ˜35%, indicating that the infused CO2 wassupersaturated into the buffered algae slurry. However, none of theinitial operating test conditions, 80-150 psi (shear influent), 80-105psi (CO2), 0 psi back pressure to the shear unit caused any significantcell disruption. Others have reported robust mechanical (blender)shear-induced algae lysis for algae exposed to the shearing forces for20 minutes. For the current tests, the shearing mechanism differs inthree important ways: algae slurry flows through an inline dual-stageparticle distribution rotor, CO2 is directly fed into the process flowstream, and exposure to shear forces in this system is 1-2 seconds.Given the short exposure time and microscopic size of the individualcells (3-5 μm), Following an initial evaluation, the culture wasre-processed through the unit a second time (2^(nd) pass) and sampleswere again imaged immediately. Although not significant, some celldisruption was visualized by the presence of additional cell debris anda 4% decline in cell number compared to controls. A 3^(rd) pass of theslurry through the system did not cause additional cell disruption,suggesting that additional processing beyond a single pass may not beuseful (for Chlorella).

Because the slurry is supersaturated with CO2 as it is processed throughthe system, and excess CO2 is lethal to many species of algae, theeffects of prolonged exposure to excess CO2 were likewise evaluated. Anadditional 60-minute incubation following processing revealed strikingdifferences between these samples and freshly processed or controlsamples. Significantly more cellular debris was visualizedmicroscopically and on a macroscopic scale, the color of the 1st passculture changed from a bright kelly green to a greenish-gray. These dataindicate not only cellular compromise, but that the phytol tails ofchlorophyll molecules within the slurry had been cleaved, changing theoverall optical character of the slurry. This effect was furtherexaggerated after a 120 minute waiting period (FIG. 13).

These tests were repeated with thawed (non-viable) algae concentrate(14% w/v) with very different results. There was no increased cellulardebris visualized following either the 1st and 2nd pass, nor was thereany increased cell disruption following an additional 60 m or 120 mincubation period. Together, these data suggest that the increased celldisruption following additional incubation in the supersaturated CO2slurry was caused by the chemical actions of excess CO2 rather thanmechanical shearing. Although CO2-induced cell disruption appears to bea somewhat effective method to compromise a small population of viableChlorella, the optical changes that occurred with this process alsosuggest that unwanted chemical degradation to targeted co-products mayalso be occurring.

As a means to increase the CO2 cavitation effects within the shear unit,the back pressure to the shear unit was incrementally increased from0-80 psi. The shear unit influent pressure was held at 100 psi and theCO2 gas pressure to the shear unit was likewise held at 100 psi. At 0psi back pressure to the shear unit, the slurry exiting the process flowcollection nozzle could be described as “sputtering and spitting” andminimal lysis was observed. Applying back pressure to the unit (20, 40,60 and 80 psi) caused both decreased sputtering and increased celldisruption, with the greatest effects at 20 psi and 40 psi (FIG. 14). Atthese back pressures, fluid flow exiting the collection nozzle wasrestored to a consistent stream. Cellular debris was estimated to be theresult of −30% cell lysis, and was quantified by cell counts to be 27%.Following an additional 2 h incubation period, total cell lysisincreased to 73%.

Summary of CO2-assisted High Shear Algae Cell Disruption. The effects ofall fluid and gas flow variables tested through the mechanical shearing(15,000 rpm) unit on viable dilute Chlorella cells were negligible.Although immediate cell disruption was not apparent, viable Chlorellacells that were further incubated in the processed supersaturated CO2slurry were disrupted in a time-dependent manner. Cell disruption causedby an extended exposure to excess CO2 caused additional chemicalreactions that negatively impact chlorophyll. Applying a back pressureof 20-40 psi to the shear unit enhanced the immediate effects of theCO2-assisted high shear process.

Diatomaceous Earth+CO2-Assisted High Shear Algae Cell Disruption.

Diatomaceous earth consists of fossilized remains of diatoms, a type ofhard-shelled (silica) algae, and is used commercially as a mildabrasive. Abrasive materials that are similar in size to the individualalgae cells (˜5 μm) can enhance cell shearing. As part of the testingmatrix, 0.01-1% w/v of diatomaceous earth (7-10 μm) was homogenized intoto the algae slurry prior to entering the process stream. The rotationalspeed of the shear unit was also increased from 15,000 rpm to 26,000rpm. A matrix of tests was conducted, with 100 psi influent, 100 psi gaspressure, and 40 psi back pressure to the shear unit yielding the bestresults. Initially, a 1% (w/v) solution was added to the algae slurryand processed through the system. This caused the system, including theplumbing and the shear unit rotor shaft to clog. Following manualcleaning, the concentration of diatomaceous earth was reduced to 0.01%(w/v). The addition of 0.01% DE to the algae slurry processed under thematrix test conditions yielded similar cell lysing results as controlswith no DE. However, the Chlorella cells that were originally present inclusters were separated into single cells following a first pass throughthe shear unit. A second pass of the 0.01% DE SSCO2 algae slurry throughthe system did not improve immediate cell rupturing. When theconcentration of DE was increased to 0.1%, the lysing efficiency from asingle pass improved significantly. Like the result using a 0.01% DEsupplement, clusters of algae cells were separated into single cells andthe percentage of lysed cells following a single pass was 54%. Further,an oily sheen was detected on the surface of the processed slurry,indicating the release of intracellular lipids from the algae cellswithin the process slurry. This result could not be repeated, however,as the system clogged repeatedly using a DE concentration of 0.1%. This54% increase in lysing may have been a single result of the accumulatedDE in the system, and when the system was cleaned, this effect was lost.

Summary of Diatomaceous Earth+CO2-Assisted High Shear Algae CellDisruption.

Increasing the rotational speed of the shear unit from 15,000 rpm to26,000 rpm (maximum) did not cause additional cell disruption overcontrols (15,000 rpm, 100 psi influent, 100 psi CO2, 40 psi backpressure). Addition of 0.01% diatomaceous earth to the process slurrycaused algae clusters to separate, but did not increase cell disruptionunder any condition over controls following two passes through thesystem. Addition of 0.1% diatomaceous earth to the process slurry causeda 24% increase in cell disruption (54%) over controls processed withoutDE (30%), likely due to excess DE in the system.

0.01% Diatomaceous Earth (DE)+CO2-Assisted High Shear (SSCO2)+ColloidMill (CM) Algae Cell Disruption.

A colloid mill is a machine that is used to reduce the particle size ofa solid in suspension in a liquid, or to reduce the droplet size of aliquid suspended in another liquid. Colloid mills work on therotor-stator principle: a rotor turns at high speeds (2000-18000 RPM).The resulting high levels of hydraulic shear applied to the processliquid disrupt structures in the fluid. Colloid mills are frequentlyused to increase the stability of suspensions and emulsions, but canalso be used to reduce the particle size of solids in suspensions.Higher shear rates lead to smaller droplets (˜1 μm) that are moreresistant to emulsion separation. For the current application, the goalwas to break ˜3-10 μm-sized algae into fragments and release commercialco-products (oil) into the surrounding medium. Therefore, an MK/2004Colloid Mill was introduced as an additional shearing mechanismdownstream of the CO2-assisted high shear unit. Primary factors thatinfluence cell shearing include the gap distance between rotors and therotation speed. Initially, the rotational speed was set to 3160 rpm andthe gap was set to 0.208 mm. The pressure of the slurry entering theshear unit was set to 100 psi, CO2 gas pressure was set to 95 psi, andthe back pressure to the shear unit was set to 40 psi. These conditionsproduced a smooth fluid flow exiting the sample collection nozzle andthe lowest pH of the processed slurry. Process controls included algaeslurries without DE, and slurries with 0.01% DE but processed throughthe colloid mill in the “off” mode. In this case, clusters of Chlorellacells were again reduced to single cells and the reduction in cell countwas ˜35%. When the colloid mill gap was decreased to 0.104 mm and no DEwas added to the process slurry, lysing improved by 5%. However, when0.01% DE was added to the process stream with a colloid mill gap widthat 0.104 mm, widespread debris was visualized and the total number ofcells decreased by 81% compared to unprocessed controls from the samebatch slurry (FIG. 15). The total intracellular oil available withinthis batch of algae slurry was calculated to be 16.7%. A small amount ofoil can often be detected in the extracellular medium as a result of thesolvent sweep method to recover lipids in the process medium. In thiscase, the percentage of oil in the extracellular medium was calculatedto be 1.8%.

Following the first pass of the slurry plus 0.01% DE through thehigh-shear unit and colloid mill with the gap adjusted to 0.104 mm, thepercentage of extracellular oil increased to 12.2%. In other words, thisprocess (0.01% Diatomaceous Earth (DE)+CO2-assisted High Shear(SSCO2)+Colloid Mill (CM), i.e., DESSCO2CM process) caused the releaseof 62% of the intracellular oil into the extracellular medium, some ofwhich could be observed at the surface of the processed slurry (FIG.16). The difference between cell lysis (81%) and lipid release (62%) maybe explained in part by noting the species of lipids that were madereadily available by the current processing parameters. Non-GMOChlorella triglycerides (˜2-20%) are typically packaged within cells aseasily accessible lipid bodies whereas phospholipids (30-85%) areembedded within membranes and can be resistant to recovery by sweepsolvents. The release of 62% of total lipids indicates that the currentprocess method significantly disrupts cellular membranes containingphospholipids allowing their recovery from a single solvent sweep.

These tests were conducted at a bench scale. Although up to 81% of algaecells were disrupted, some operational challenges were noted. Under allof the combinations of parameters tested, the addition of diatomaceousearth appeared to be necessary to increase lysing efficiency.

Summary of Diatomaceous Earth+CO2-Assisted High Shear+Colloid Mill AlgaeCell Disruption.

The addition of the downstream colloid mill with the gap set to 0.104 mmimproved algae cell lysis by ˜50%, for a total of 81% of cells ruptured.The following parameters yielded 81% cell lysis and 62% lipid release:0.01% DE; IKA Magic high shear unit, 26,000 rpm; colloid mill, 3160 rpm,0.104 mm gap; 100 psi influent pressure (to the shear unit); 95 psi CO2gas pressure (to the shear unit); 40 psi back pressure (to the shearunit). DE accumulates within the small diameter tubing in the systemover time and decreases fluid flow and lysing efficiency, and eventuallyleads to severe blockage that requires manual cleaning. Larger scalesystems may be less susceptible to DE accumulation. Given the low energyrequirements for this system, this process could significantly improvethe energy balance for production of algal biofuels, especially if itcan be synergistically combined with a vacuum to efficiently harvestreleased algal cell lipids.

Example 3 Gas-Assisted High Shear Cell Lysing

Flow-through algae cell disruption testing was demonstrated in Example 2using a CO2-infused high-speed, high-pressure rotor-stator homogenizer(HSPH) and colloid mill homogenizer (CMH). The testing showed that theinfusion of pressurized CO2 into the HSPH and immediate exposure to theCMH are unique and important drivers of the cellular disruptionefficiency. A proposed mechanism states that the rapid infusion of CO2into the algal cells causes intracellular disruption and cellularswelling, and the immediate subsequent exposure to shear forcessignificantly enhances cellular disruption and product recovery.Although energy balance was not examined, the algae were compromised ina single pass, and therefore, the required energy input into the systemis expected to be far less than current non-CO2 shearing technologiesthat require multiple passes.

The CO2-assisted shearing system was likewise tested using yeast as thebio-feedstock. After replacing CO2 with O2, significant yeast lysis wasobserved.

Materials and Methods. Yeast Biomass Production.

A genetically engineered strain of yeast, Yarrowia lipolytica, providedby Dr. Hal Alper, University of Texas. Baker's yeast, Saccharomycescerevisiae were fermented in 37° C. dH2O supplemented with 20% D-glucoseand 2% bacto yeast extract within 2 L sterilized glass bioreactors.Culture density was recorded by absorbance at 600 nm. Cultures near theend of the exponential phase of growth were used for testing.

CO2-Assisted High Shear Algae Cell Disruption Set Up and Test Matrix.

The process flow for CO2-assisted high shear yeast cell disruption isshown in. Biomass slurry sample flow was initiated as follows: the fluidflow pressure of dilute or concentrated yeast slurry through the systemwas initiated at 100 psi. The high shear unit was then turned on andmaintained at a rotational speed of 26,000 rpm. Diffused CO2 wasintroduced into the system at the shear unit an initial pressure of 80psi, then adjusted to 95 psi to reduce sample sputtering from thecollection nozzle. The colloid mill was then activated. In some testcases, the back pressure to the shear unit was increased in incrementsof 20 psi from 0 psi to 40 psi. Once the target test parameters weremet, processed samples were collected in Erlenmeyer flasks forsubsequent cell disruption analyses.

O2-Assisted High Shear Yeast Cell Disruption Set Up.

The process flow for O2-assisted high shear yeast cell disruption wasidentical to the CO2-assisted high shear system except that the CO2 wasreplaced with O2 gas infusion.

Quantification of Cell Disruption.

The effectiveness of each treatment was qualitatively visualized bybrightfield microscopy (10×-40×), and quantified by measuring thefraction of physically disrupted cells. ImageJ, a public-domain imageprocessing and analysis software, was used to ensure consistency in cellcounting by minimizing variability in analysis between samples.Measurements of the total area occupied by cells were used to verify theintact cell counts. This was particularly useful for samples with cellclumping, typically observed in samples that had been homogenized athigher pressures. All imaging was performed using an AmScope B120C-E1Siedentopf Binocular Compound Microscope with a 1.3 MP digital camera.Cell counts were compared to unprocessed (flowed through the system butno shear, mill, +/−CO2 or O2) yeast from the same batch.

Results and Discussion.

CO2-assisted High Shear Yeast Cell Disruption. In order to identify theoperating conditions that maximized yeast cell disruption, a test matrixin which individual parameters were varied one at a time was created.Yeast was initially processed through the CO2-assisted high shear,high-pressure homogenization (HSPH) system using the parameters thatwere previously observed to maximize algae cell disruption (shear unitspeed, 26,000 rpm; colloid mill, 3160 rpm, gap 0.14 mm; 100 psi shearunit influent; 95 psi CO2 to the shear unit; 40 psi back pressure to theshear unit). No diatomaceous earth was used in the yeast processtesting.

Despite using the same conditions that caused ˜80% of processedChlorella cells to lyse, no significant cell lysis was observed forprocessed yeast cells after one, two, or three processing passes.Increasing the back pressure to the shear unit likewise had nosignificant effect on yeast cell lysis. The differences between algaeand yeast cellular respiration requirements (CO2 vs O2) may explain thisresult. Algae readily take up CO2 in the form of CO2 gas or dissolvedcarbonate and release O2 as a byproduct of cellular respiration. Incontrast, yeast preferentially take up O2 by gradient diffusion acrossits membrane and releases CO2 as a byproduct of cellular metabolism.When there are two gases separated by a permeable membrane (like thecell/plasma membrane), the gas will move across the membrane from thehigh concentration environment, in this case the gas-infused shear unit,to the low concentration of that gas (the intracellular compartment)until the concentrations on each side equalize. As long as there isalways a lower concentration of oxygen inside the cell than outside thecell, oxygen will continuously diffuse into the cell. Isenschmid et al.(1995) reported that yeast exposed to 60 bar (870 psi) CO2 for 15minutes were not lysed; rather the yeast cells appeared intact but wererendered non-viable likely due to CO2 toxicity. For the current tests,the goal was cellular destruction that facilitated lipid extraction andrecovery, and therefore additional testing using CO2 as the infusion gaswas discontinued, and replaced with O2 gas.

Substituting O2 as the gas infused into the shear mechanism waspredicted to have advantages over CO2-assisted shearing. Firstly, yeastcells preferentially take up O2 gas. Because yeast efficientlymetabolize O2, the concentration of O2 within the intracellularcompartment remains low and the concentration gradient for O2 diffusioninto yeast cells is comparatively large. Second, the supersaturatingconcentration of O2 within the shear unit forces excess O2 into thecells. Excess O2 intake within yeast leads to the generation of reactiveoxygen species within the intracellular compartment, which in turn leadsto cell expansion and eventually cell death (FIG. 17). The expandedmembranes of O2-supersaturated yeast cells were predicted to have lesslocalized tensile strength and therefore more susceptible to shearingforces.

O2-Assisted High Shear Yeast Cell Disruption.

Yeast were processed through the O2-HSPH-CM system, with minormodifications to the operating parameters (90 psi (from 100 psi) to theshear unit and 85 psi (from 95 psi) O2) to accommodate differences inslurry viscosity and smooth process flow, then visualized at 40× andquantified for changes in cell density (FIGS. 18-19). Clusters of yeastcells observed in unprocessed control samples appeared to be broken upin processed samples, especially those that were exposed tosupersaturating O2. Cell density was reduced in the samples where 20 psior 40 psi back pressure to the shear unit was applied, −12% and −9%respectively. No cell reduction was observed in samples that were notexposed to supersaturating O2. Higher magnification microscopy revealedthat the cell membranes of yeast exposed to excess O2 visually appearedthinner and the intracellular compartment somewhat more opaque. Despitethese morphological changes, the cells largely remained intact. After 30minutes, the yeast slurry was processed through the O2-HSPH-CM a secondtime. Microscopic inspection and cell counting revealed no significantchanges in either morphology or cell density, indicating that a delayedsecond pass had no significant effects on the process slurry.

Once yeast cells were exposed to supersaturating O2, it was predictedthat the cells would swell and cellular membranes would be susceptibleto shear forces. In addition, SSO2 causes the generation of ROS andsubsequent cell death. The 30 minute delay between process passes wasnot long enough to cause cell death (typically occurs in 4-6 hours), butmay have provided enough time for cells to initiate defense mechanismsthat restore cellular integrity, albeit futile in the end. To test thishypothesis, yeast slurry was processed through the O2-HSPH-CM using thesame operating parameters as the previous test, then immediatelyprocessed a second time through the system. An immediate second processpass caused significant cellular disruption, visualized as widespreaddebris and quantified by cell counts. The cell density of the slurry wasreduced by 36% (20 psi BP) and 34% (40 psi BP) compared to controls thatwere circulated through the system but not exposed to O2 or shearforces.

To characterize the effects of multiple passes through the O2-HSPH-CMsystem, a follow-up test whereby the slurry was continually circulatedthrough the system for 5 minutes was conducted. Each process pass wascalculated to require 1.2 minutes. Therefore, an average yeast cellcirculating through the system for 5 minutes was exposed to ˜4 passesthrough the system. Widespread debris was again visualized in samplesthat were exposed to O2-HSPH-CM (FIGS. 20C and 20D), and a furtherreduction in cell density was likewise documented, −47% (20 psi BP) and−53% (40 psi BP) compared to unprocessed controls (FIG. 20A).Interestingly, O2 appears to be a critical feature for significant yeastdisruption through this system. Like the previous tests using shearforces, yeast cells clusters were reduced to single cells, however, thereduction in slurry density was minimal (˜15%) (FIG. 20B). Together,these data indicate that shear forces are effective in separatingclusters of cells and disrupting O2 saturated cells, and that effectivecell lysis occurs immediately following cellular compromise by excessintracellular O2. Additional testing is required to optimize the lysingperformance of the O2-high shear processing unit-colloidal mill systemand determine whether the effects are species specific.

SUMMARY

Despite substantial progress in the development of cell factories forthe production of advanced biofuels, there is still need for furtherimprovement in the production capacity of these cell factories and intechnologies that extract and recover the synthesized fuel products.Engineered yeast cells are an attractive platform for renewable fuelproduction, but due to the high tensile strength of yeast membranes,cost-effective lysis of these cells for recovery their fuel products haslimited the scalability of this platform. The method as discussed hereinwhereby O2 is supersaturated into the process stream within a rotationalshear device coupled with a colloid mill was shown to rupture >50% ofyeast cells within 5 minutes with no added heat or chemical agents.Additional circulation through the system and minor changes to operatingprocedures will likely increase lysing efficiency further. Unlike simpleexternal shearing forces created by commercial homogenizers, thecellular disruption caused by the O2-high shear processingunit-colloidal mill system is thought to be the result of a differentmechanism, namely by creating intracellular disarray, membrane swellingand loss of structural integrity that increases the cell'ssusceptibility to subsequent shear forces.

Example 4 Demonstration of High Shear Cell Lysing

As shown previously, the gas-assisted high shear process validated thatthe system is capable of 1) supersaturating algae growth media with CO2which leads to enhanced CO2 consumption and bioremediation, 2) rupturinga significant fraction of Chlorella vulgaris algae or Saccharomyces sp.yeast following a single process pass. The process flow path includes agas-assisted 3-stage high shear unit followed by a colloid mill.Previous cell lysis tests suggested that the first exposure togas-assisted high shear causes cells within the slurry to take up excessgas and swell, a condition that compromises cell integrity. A secondexposure to shear while the cells remain compromised is sufficient torupture up to 92% of algal cells and 52% of yeast cells. The primaryfocus of the last set of tests was to identify process flow pressuresthat optimized cell lysis. Previous tests indicated that a high shearunit rotational speed greater than 15,000 rpm was required to maximizealgae cell lysis. Therefore, the rotational speed was increased to26,000 rpm, a condition that resulted in enhanced algae cell lysis. Therotational speed of the high shear unit was likewise held constant at26,000 rpm for subsequent yeast lysis testing. For the current tests, weinvestigated the effect of O2-assisted high shear speed vs. cell lysis.

Materials and Methods. Yeast Biomass Production.

Baker's yeast, Saccharomyces cerevisiae, were inoculated into 37° C.dH2O supplemented with 20% D-glucose and 2% bacto yeast extract within 2L sterilized glass bioreactors supplemented with constant ambientaeration. Culture density was recorded by absorbance at 600 nm. Thecultures were scaled up within 20 L glass carboys followed by transferto 90 L vertical airlift photobioreactors. Cultures near the end of theexponential phase of growth were used for testing.

O2-Assisted High Shear Algae Cell Disruption Set Up and Test Matrix.

The process flow for O2-assisted high shear yeast cell disruption isshown in FIG. 21. Yeast slurry sample flow was established in rapidsuccession as follows: the fluid flow pressure of dilute yeast slurrythrough the system was initiated at 100 psi then adjusted to 90 psi. Thehigh shear unit was then turned on and maintained at a rotational speedof 3,000-26,000 rpm as indicated. Concurrently, diffused O2 wasintroduced into the system at the shear unit an initial pressure of 100psi, then adjusted to 85 psi to reduce sample sputtering from thecollection nozzle. Control runs were evaluated by running the slurryflowed through the system The back pressure to the shear unit wasadjusted to 40 psi. Once the target test parameters were met, a timerwas started. Processed samples were collected 2 minutes later, or afterapproximately 2 passes through the system, in Erlenmeyer flasks forsubsequent cell disruption analyses.

Quantification of Cell Disruption.

The effectiveness of each treatment was qualitatively visualized bybright field microscopy (40×), and quantified by measuring the fractionof physically disrupted cells (Spiden, 2013). ImageJ, a public-domainimage processing and analysis software, was used to ensure consistencyin cell counting by minimizing variability in analysis between samples.Measurements of the total area occupied by cells were used to verify theintact cell counts. This was particularly useful for samples with cellclumping, typically observed in samples that had been homogenized athigher pressures. All imaging was performed using an AmScope B120C-E1Siedentopf Binocular Compound Microscope with a 1.3 MP digital camera.Cell counts were compared to unprocessed (flowed through the system butno shear, mill) yeast from the same batch.

Results and Discussion. Effect of Shear Speed on O2-Assisted High ShearYeast Cell Disruption.

A test matrix in which the operational shear speed of the IKAmulti-stage high shear unit was adjusted from 0 rpm (negative control)to 26,000 rpm (see Table 1).

TABLE 1 Test matrix of O2-assisted high shear speed vs. yeast celllysis. Back pressure colloid mill % reduction in circulation time pumpO2 to shear colloid mill clearance cell number (in min) pressurepressure unit shear unit rotor gap (in (compared to (1 pass = 60 s)(psi) (psi) (psi) speed speed (Hz) mm) control) generate sufficient (100L) yeast culture start-up procedure 2 90 85 20 26000 3160 0.104 55.2 290 85 20 22000 3160 0.104 56.8 2 90 85 20 18000 3160 0.104 15.1 2 90 8520 14000 3160 0.104 6.2 2 90 85 20 10000 3160 0.104 6.6 2 90 85 20 60003160 0.104 0 2 90 85 20 3000 3160 0.104 1.5 2 90 85 20 0 3160 0.104 0shut down procedure start-up procedure 2 90 85 20 26000 0 0.104 49.1 290 85 20 22000 0 0.104 47.9 2 90 85 20 18000 0 0.104 17.8 2 90 85 2014000 0 0.104 16 2 90 85 20 10000 0 0.104 9.2 2 90 85 20 6000 0 0.1045.4 2 90 85 20 3000 0 0.104 4.3 2 90 85 20 0 0 0.104 0 shut downprocedure report total lab time (min)

Initially, yeast slurry was circulated through the system while both thehigh shear unit and colloid mill were not activated. Samples werecollected following a 2 minute circulation and the cell density wasevaluated and established as 100%. For the first set of shear speed vs.cell disruption tests, the colloid mill operational speed was held at3160 rpm and the mill gap was held at 0.104 mm, reflecting the optimizedspeed and gap distance identified in previous tests while theoperational speed of the high shear unit was incrementally varied witheach process run.

FIG. 22 shows a linear relationship between lower shear speeds (3000rpm-18,000 rpm) and cell disruption. The effect of the downstreamcolloid mill was minimal. Cell disruption was significantly improvedwith high shear speeds from 18,000 rpm through 26,000 rpm. Again, theeffect of the colloid mill was minimal under this scenario. This resultdiffers from previous testing where the colloid mill increased celldisruption after a single pass. Previous testing showed that after asingle pass through the high shear unit at 26,000 rpm, ˜70% of yeastcells appeared swollen and stressed, but intact. A subsequent exposureto a shearing mechanism (colloid mill, 3160 rpm, 0.104 mm gap) caused asignificant fraction of these compromised cells to lyse. For the currenttests, the slurry was circulated for 2 minutes and the slurry wasexposed to O2-assisted high shear at least twice, or both the high shearunit plus the colloid mill for 2 minutes for a total of −4 passesthrough a shearing mechanism. These data indicate that multipleexposures to O2-assisted high shear at rotational speeds greater than18,000 rpm is sufficient to rupture ˜50-56% of yeast cells within theprocess stream and that the addition of another downstream shearmechanism may not be necessary.

While preferred embodiments of the invention have been shown anddescribed, modifications thereof can be made by one skilled in the artwithout departing from the spirit and teachings of the invention. Theembodiments described herein are exemplary only, and are not intended tobe limiting. Many variations and modifications of the inventiondisclosed herein are possible and are within the scope of the invention.Where numerical ranges or limitations are expressly stated, such expressranges or limitations should be understood to include iterative rangesor limitations of like magnitude falling within the expressly statedranges or limitations (e.g., from about 1 to about 10 includes, 2, 3, 4,etc.; greater than 0.10 includes 0.11, 0.12, 0.13, and so forth). Use ofthe term “optionally” with respect to any element of a claim is intendedto mean that the subject element is required, or alternatively, is notrequired. Both alternatives are intended to be within the scope of theclaim. Use of broader terms such as comprises, includes, having, etc.should be understood to provide support for narrower terms such asconsisting of, consisting essentially of, comprised substantially of,and the like.

Accordingly, the scope of protection is not limited by the descriptionset out above but is only limited by the claims which follow, that scopeincluding all equivalents of the subject matter of the claims. Each andevery claim is incorporated into the specification as an embodiment ofthe present invention. Thus, the claims are a further description andare an addition to the preferred embodiments of the present invention.The disclosures of all patents, patent applications, and publicationscited herein are hereby incorporated by reference, to the extent theyprovide exemplary, procedural or other details supplementary to thoseset forth herein.

What is claimed is:
 1. A method of processing a medium containing algaemicroorganisms to produce algal oil and by-products, comprisingproviding the medium containing algae microorganisms; passing saidmedium through a rotor-stator high shear device; disintegrating cellwalls of and intracellular organelles in the algae microorganisms torelease algal oil and by-products; and removing the algae medium from anoutlet of the high shear device.
 2. The method of claim 1 wherein saiddisintegration is enhanced by a penetrating gas capable of permeatingthe cell wall.
 3. The method of claim 2 wherein said enhancement isaccomplished by super-saturation of the penetrating gas in the medium orincreased gas pressure in a vessel.
 4. The method of claim 2 whereinsaid penetrating gas is different from the gas produced by the cellduring respiration.
 5. The method of claim 1 wherein the mediumcontaining algae microorganisms is de-watered at least partially beforethe medium is passed through said high shear device.
 6. The method ofclaim 5 wherein a solvent is added to the at least partially de-wateredmedium before the medium is passed through said high shear device. 7.The method of claim 6 wherein said solvent is a gas comprising carbondioxide or air or oxygen or nitrogen; or a liquid comprising an alcoholor hexane or vegetable oil and/or animal fat or tallow.
 8. The method ofclaim 1 further comprising separating algal oil and byproducts from thealgae medium removed from the high shear device.
 9. The method of claim8 further comprising converting the algal oil to biodiesel.
 10. Themethod of claim 1 further comprising producing said medium containingalgae microorganisms, comprising super-saturating a liquid with carbondioxide in a second rotor-stator high shear device operating at a shearrate of greater than 1,000,000 s⁻¹; feeding said carbon dioxidesupersaturated liquid and a nutrient source to algae microorganisms andoptionally bacteria; allowing the algae microorganisms to grow byconsuming carbon dioxide and the nutrient; and generating said mediumcontaining algae microorganisms.
 11. The method of claim 10 wherein saidnutrient source comprises municipal waste; sewage waste; paper pulp;chemical and petrochemical; vegetable including grain, sugar; farmdischarge; animal farm discharge including beef, pork, poultry; canningdischarge, fishing discharge; farming discharge; food processingdischarge.
 12. The method of claim 11 wherein said nutrient source ispretreated to eliminate undesirable pathogens via gas-assisted highshear lysing of pathogen cells or pretreated using high shear toincrease the bio-availability of nutrient in the nutrient source. 13.The method of claim 10 wherein said algae and/bacteria are geneticallymodified.
 14. The method of claim 10 wherein said bacteria cause thebreakdown of said nutrient source to promote algae growth.
 15. A systemcomprising a rotor-stator high shear device configured to process amedium containing algae microorganisms to produce algal oil andby-products, wherein said high shear device is operated to disintegratecell walls of and intracellular organelles in the algae microorganismsto release algal oil and by-products, wherein said high shear devicecomprises an inlet to take in said medium containing algaemicroorganisms and an outlet for the algae medium to be removed from thehigh shear device.
 16. The system of claim 15 comprising at least tworotor-stator high shear devices fluidly connected in series to processsaid medium containing algae microorganisms and disintegrate cell wallsof and intracellular organelles in the algae microorganisms to releasealgal oil and by-products.
 17. The system of claim 15 further comprisinga separation system configured to separate algal oil and by-productsfrom the medium.
 18. The system of claim 15 further comprising aconversion system configured to convert algal oil to biodiesel.
 19. Thesystem of claim 15 further comprising a tank or pond configured to growalgae containing algae microorganisms and optionally bacteria; anutrient source consumable by said algae microorganisms and optionallybacteria; another rotor-stator high shear device configured to processcarbon dioxide in a liquid operating at a shear rate of greater than1,000,000 s⁻¹ to form a carbon dioxide super-saturated liquid stream andfeed said stream into the tank/pond for algae growth; and a fluid lineconfigured to extract a medium containing algae from the tank or pondand send the medium to the rotor-stator high shear device configured toprocess said medium.
 20. The system of claim 19 is modular and isoptionally integrated with an existing facility.